Jasmonate derivative compounds pharmaceutical compounds and methods of use thereof

ABSTRACT

The present invention provides novel jasmonate derivative compounds, methods for their preparation, pharmaceutical compositions including such compounds, and methods of using these compounds and compositions, especially as chemotherapeutic agents for treatment of cancers, especially mammalian cancers.

FIELD OF THE INVENTION

The present invention relates to the field of jasmonate derivativecompounds, methods for their preparation, pharmaceutical compositionsincluding such compounds, and methods of using these compounds andcompositions, especially as chemotherapeutic agents for treatment ofcancers, especially cancers in mammals.

BACKGROUND OF THE INVENTION

Jasmonates are a family of plant stress hormones, derived from linolenicacid by the octadecanoid pathway, and are found in minute quantities inmany edible plants. Stress hormones such as the jasmonate family, haveevolved in plants, and are released in such times of stress such asextreme UV radiation, osmotic shock, heat shock and pathogen attack toinitiate various cascades which end in appropriate responses. Examplesof members of the jasmonate family are jasmonic acid, which is crucialto intracellular signaling in response to injury, and methyl jasmonate,which causes induction of a proteinase inhibitor that accumulates at lowconcentrations in response to wounding or pathogenic attacks. Jasmonateshave been patented for a variety of uses in plant growth and cropimprovement, but have not been previously known for use in medicine. Useof jasmonates for the treatment of mammalian cancer has been disclosedin U.S. Pat. No. 6,469,061, reference to which is incorporated hereby inits entirety. In U.S. Pat. No. 6,469,061, it was shown that jasmonateswere directly cytotoxic for various types of human cancer cells derivedfrom breast, prostate, skin and blood cancers. While jasmonates eliciteddeath in human leukemic Molt-4 cells, they did not damage normallymphocytes.

Subsequent data collected similarly showed that jasmonates do not damagehealthy erythrocytes (see WO 02/080890). In U.S. Pat. No. 6,469,061, onejasmonate compound in particular, methyl jasmonate, was shown to beeffective in preventing development of lymphomas in mice. See alsoFingrut, O. and E. Flescher. 2002. “Plant stress hormones suppress theproliferation and induce apoptosis in human cancer cells”, Leukemia 16:608-616 (2002).

The pharmacological activity of jasmonate compounds makes themattractive candidates as therapeutic agents for the treatment of cancer.Because only a few jasmonate derivatives have been reported (see, forexample, Ishii et al., Leukemia, 1-7 (2004)), a need in the art existsto develop jasmonate derivative compounds that are potentchemotherapeutic drugs, with a high degree of specificity towardsmalignant cells.

The present invention addresses this need, and provides other advantagesas well.

SUMMARY OF THE INVENTION

The present invention is directed to jasmonate derivative compounds,especially those that are halogenated. Such compounds include “methyljasmonate di-bromide”, or “MJDB”, and “methyl jasmonate tetra-bromide”,or “MJTB.” These compounds are significantly more potent than the mosteffective jasmonate disclosed in U.S. Pat. No. 6,469,061, namely, methyljasmonate. Jasmonate derivatives such as MJDB and MJTB, as shown below,exert selective cytotoxicity on cancerous lymphocytes drawn frompatients, while sparing normal lymphocytes.

The present invention also includes including salts, hydrates, solvates,polymorphs, optical isomers, diastereomers, and any mixtures thereof ofthe compounds of the present invention, especially of MJDB and MJTB.

The compounds of the present invention have the formula:

-   -   wherein:    -   n is 0.1, or 2;    -   R₁ is OH, C₁ to C₁₂ alkoxy, C₁ to C₁₂ substituted alkoxy        aryloxy, O-glucosyl or imino;    -   R₂ is OH, C₁ to C₁₂ alkoxy, C₁ to C₁₂ substituted alkoxy,        O-glucosyl, oxo, alkyl or imino;    -   R₃, R₄, R₅, R₆, R₇, A, B, C, D and E are each independently H,        halogen, OH, C₁ to C₁₂ alkoxy, C₁ to C₁₂ substituted alkoxy,        aryloxy, O-glucosyl, C₁ to C₁₂ alkyl or C₁ to C₁₂ substituted        alkyl;    -   wherein R₁ and R₂, or R₁ and R₄ may form together a lactone        which is optionally substituted;    -   wherein the bonds between C₃:C₇, C₄:C₅, and C₉:C₁₀ may        independently be double bonds or single bonds;    -   provided that at least one of R₃, R₄, R₅, R₆, R₇. A, B, C, D and        E is a halogen; and provided that, if A is the only halogen in        the compound, that A is not fluoro;    -   or a derivative of said formula, wherein the derivative has at        least one of the following:    -   a lower acyl side chain at C₃ (free acid or ester or conjugate),        a keto or hydroxy (free hydroxy or ester) moiety at the C₆        carbon, or an n-pentenyl or n-pentyl side chain at C₇;    -   including salts, hydrates, solvates, polymorphs, optical        isomers, enantiomers, diastereomers, and mixtures thereof.

More specifically, the preferred compounds of the present invention(Formula I) are those where the bond between C₉ and C₁₀ is a singlebond. Other preferred compounds are where R₂ is oxo. Still otherpreferred compounds are where at least one of R₆ and R₇ is bromo, iodo,fluoro or chloro. Even more preferred are compounds where both of R₆ andR₇ are selected from bromo, iodo, fluoro or chloro. Yet more preferredare compounds where both of R₆ and R₇ are bromo.

A further preferred aspect of the invention are compounds where A, B R₆and R₇ me bromo, iodo, fluoro or chloro. Even more preferred arecompounds where A, B, R₆ and R₇ are each bromo.

Other preferred compounds of the present invention are where R₁ isalkoxy. In yet another aspect, R₃, R₄ and R₅ are each H (hydrogen). Instill another aspect, C, D and E are each H.

One of the most preferred compounds of the present invention is methyljasmonate di-bromide (MJDB). According to Formula I, MJDB is where: n is0; the bonds between C₃:C₇, C₄:C₅, and C₉:C₁₀ are single bonds; R₁ ismethoxy; R₂ is oxo; R₃, R₄, R₅, A, B, C, D and E are each H; and R₆ andR₇ are each bromo.

Another of the most preferred compounds of the present invention ismethyl jasmonate tetra-bromide (MJTB). According to Formula I, MJTB iswhere: n is 0; the bonds between C₃:C₇, C₄:C₅, and C₉:C₁₀ are singlebonds; R₁ is methoxy; R₂ is oxo; R₃, R₄, R₅, C, D and E are each H; andA, B, R₆ and R₇ are each bromo.

In other preferred compounds of the invention n is 0; the bonds betweenC₃:C₇, C₄:C₅, and C₉:C₁₀ are single bonds; R₁ is methoxy; R₂ is oxo; R₃,R₄, R₅, A, B, C, D and E are each H; and either:

e) R₆ and R₇ are each fluoro (designated as compound “MJS99”);

b) R₆ and R₇ are each iodo (designated as compound “MJS85f14”);

c) R₆ and R₇ are each chloro (designated as compound “MJS81f13”);

d) one of R₆ and R₇ is bromo and the other is hydroxy (designated ascompound “NJ-63); or

e) one of R₆ and R₇ is iodo and the other is methoxy (designated ascompound “MJS72f5).

The present invention also contemplates pharmaceutical compositions thatinclude a pharmaceutically acceptable carrier and, as an activeingredient, the compounds of the invention, as described above.Preferred compositions have as an active ingredient MJDB or MJTB.Preferably, in the pharmaceutical composition the active ingredient isdissolved in any acceptable lipid carrier. Further, in accordance with apreferred embodiment of the present invention, the compositionadditionally comprises at least one other chemotherapeutic agent.

The present invention additionally provides a method for reduction ofthe growth of cancer cells, comprising exposing the cancer cells to atherapeutically effective amount of a compound of the invention, asdescribed herein

Furthermore, the present invention provides a method for the treatmentof cancer, comprising administering to the subject a pharmaceuticalcomposition containing as an active ingredient a therapeuticallyeffective amount of the compound of the invention, as described herein.According to preferred embodiments the cancers are cancers of awarm-blooded vertebrate, more preferably a mammal, most preferably ahuman.

The cancers include carcinoma, sarcoma, adenoma, hepatocellularcarcinoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma,thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphagiosarcoma, synovioama, Ewing's tumor,leimyosarcoma, rhabdotheliosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hematoma,bile duct carcinoma, melanoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms' tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell and non-small cell lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocyoma, medulloblastoma,craniopharyngioma, ependynoma, pinealoma, retinoblastoma, rectalcarcinoma, cancer of the thyroid, head and neck cancer, brain cancer,cancer of the peripheral nervous system, cancer of the central nervoussystem, neuroblastoma, cancer of the endometrium, lymphoproliferativediseases, hematopoietic malignancies including all types of leukemia andlymphoma including: acute myelogenous leukemia, acute myelocyticleukemia, acute lymphocytic leukemia, chronic myelogenous leukemia,chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma,myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, as well asmetastases of all the above.

These and further features of the present invention will be betterunderstood in conjunction with the drawings, detailed description, andclaims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is herein described, by way of example only, withreference to the accompanying drawings, wherein:

FIG. 1 shows the cytotoxic activity of MJDB against: a) human CLLlymphocytes (labeled “Patient 1,” “Patient 2” and “Patient 3”); and b)lymphocytes from healthy donors (labeled “Normal”).

FIG. 2 shows a comparison of the level of toxicity effected by MJDB andby methyl jasmonate (MJ) on four human malignant cell lines:lymphoblastic leukemia (Molt-4), lung carcinoma (3LL), melanoma (B16)and colon carcinoma (HCT116).

FIG. 3 shows the cytotoxic activity of MJDB, against cells expressingeither wild type or mutant p53.

FIG. 4 shows a comparison of the effect of MJDB and MJ on ATP levels inMolt-4 leukemia cells.

FIG. 5 shows the cytotoxic activity of MJTB against different celllines: Molt4 (leukemia), D122 (lung carcinoma), B16 (melanoma) andB16MDR (melanoma exhibiting multidrug resistance.

FIG. 6 compares the cytotoxic activity of MJTB and MJ. Cytotoxicity iscalculated as % of control untreated cultures, mean±SE. n=3. *** denotesP<0.001 comparing the effects of MJ and MJTB.

FIG. 7 compares the effect of MJTB on normal lymphocytes versus leukemiccells. * denotes P<0.05, ** denotes P<0.01, *** denotes P<0.001comparing the effects of MJTB on leukemic versus normal lymphocytes.Cytotoxicity was calculated as % of control untreated cultures, mean±SE.n=3.

FIG. 8 shows the cytotoxicity of other MJ derivatives of the inventionagainst leukemia cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel jasmonate derivatives, compositionscomprising these compounds and methods of using the compositions in thetreatment of cancer.

The compounds of the present invention have the formula:

-   -   wherein:    -   n is 0, 1, or 2;    -   R₁ is OH, C₁ to C₁₂ alkoxy, C₁ to C₁₂ substituted alkoxy,        aryloxy, O-glucosyl or imino;    -   R₂ is OH, C₁ to C₁₂ alkoxy, C₁ to C₁₂ substituted alkoxy,        O-glucosyl, oxo, alkyl or imino;    -   R₃, R₄, R₅, R₆, R₇, A, B, C, D and E are each independently H.        halogen, OH, C₁ to C₁₂ alkoxy, C₁ to C₁₂ substituted alkoxy,        aryloxy, O-glucosyl, C₁ to C₁₂ allyl or C₁ to C₁₂ substituted        alkyl;    -   wherein R₁ and R₂, or R₁ and R₄ may form together a lactone        which is optionally substituted;    -   wherein the bonds between C₃:C₇, C₄:C₅, and C₉:C₁₀ may        independently be double bonds or single bonds;    -   provided that at least one of R₃, R₄, R₅, R₆, R₇, A, B, C, D and        E is a halogen; and provided that, if A is the only halogen in        the compound, that A is not fluoro:    -   or a derivative of said formula, wherein the derivative has at        least one of the following:    -   a lower acyl side chain at C₃ (free acid or ester or conjugate),        a keto or hydroxy (free hydroxy or ester) moiety at the C₆        carbon, or an n-pentenyl or n-pentyl side chain at C₇;    -   including salts, hydrates, solvates, polymorphs, optical        isomers, enantiomers, diastereomers, and mixtures thereof.

More specifically, the preferred compounds of the present invention(Formula I) are those where the bond between C₉ and C₁₀ is a singlebond. Other preferred compounds are where R₂ is oxo. Still otherpreferred compounds are where at least one of R₆ and R₇ is bromo, iodo,fluoro or chloro. Even more preferred are compounds where both of R₆ andR₇ are selected from bromo, iodo, fluoro or chloro. Yet more preferredare compounds where both of R₆and R₇ are bromo.

A further preferred aspect of the invention are compounds where A, B, R₆and R₇ are bromo, iodo, fluoro or chloro. Even more preferred arecompounds where A, B, R₆ and R₇ are each bromo.

Other preferred compounds of the present invention are where R₁ isalkoxy. In yet another aspect, R₃, R₄ and R₅ are each (hydrogen). Instill another aspect, C, D and E are each H.

One of the most preferred compounds of the present invention is methyljasmonate di-bromide (MJDB). According to Formula I, MJDB is where: n is0; the bonds between C₃:C₇, C₄:C₅, and C₉:C₁₀ are single bonds; R₁ ismethoxy; R₂ is oxo; R₃, R₄, R₅, A, B, C, D and E are each H; and R₆ andR₇ are each bromo.

Another of the most preferred compounds of the present invention ismethyl jasmonate tetra-bromide (MJTB). According to Formula I, MJTB iswhere: n is 0; the bonds between C₃:C₇, C₄:C₅, and C₉:C₁₀ are singlebonds; R₁ is methoxy; R₂ is oxo; R₃, R₄, R₅, C, D and E are each H; andA, B, R₆ and R₇ are each bromo.

In other preferred compounds of the invention ii is 0; the bonds betweenC₃:C₇, C₄:C₅, and C₉:C₁₀ are single bonds; R₁ is methoxy; R₂ is oxo; R₃,R₄, R₅, A, B, C, D and E are each H; and either:

a) R₆ and R₇ are each fluoro (designated as compound “MJS99”);

b) R₆ and R₇ are each iodo (designated as compound “MJS85f14”);

c) R₆ and R₇ are each chloro (designated as compound “MJS81f13”);

d) one of R₆ and R₇ is bromo and the other is hydroxy (designated ascompound “NJ-63); or

e) one of R₆ and R₇ is iodo and the other is methoxy (designated ascompound “MJS72f5).

The present invention also contemplates pharmaceutical compositions thatinclude a pharmaceutically acceptable carrier and, as an activeingredient, the compounds of the invention, as described above.Preferred compositions have as an active ingredient MJDB or MJTB.Preferably, in the pharmaceutical composition the active ingredient isdissolved in any acceptable lipid carrier. Further, in accordance with apreferred embodiment of the present invention, the compositionadditionally comprises at least one other chemotherapeutic agent.

The present invention additionally provides a method for reduction ofthe growth of mammalian cancer cells, comprising applying to the cancercells a therapeutically effective amount of a compound of the invention,as described herein

Furthermore, the present invention provides a method for the treatmentof cancer in warm-blooded vertebrates, especially cancer in mammals,comprising administering to the subject a pharmaceutical compositioncontaining as an active ingredient a therapeutically effective amount ofthe compound of the invention, as described herein. The term “mammals”includes non-human mammals and humans.

It is to be understood that whenever the terms “treating or inhibiting amalignant cell proliferative disease or disorder”, “treating orinhibiting a non-solid cancer”, “treating or inhibiting a tumor” areused herein in the description and in the claims, they are intended toencompass tumor formation, primary tumors, tumor progression or tumormetastasis.

The term “reduction of growth” in relation to cancer cells, in thecontext of the present invention refers to a decrease in at least one ofthe following: number of cells (due to cell death which may be necrotic,apoptotic or any other type of cell death or combinations thereof) ascompared to control; decrease in growth rates of cells, i.e. the totalnumber of cells may increase but at a lower level or at a lower ratethan the increase in control; decrease in the invasiveness of cells (asdetermined for example by soft agar assay) as compared to control evenif their total number has not changed; progression from a moredifferentiated cell type to a less differentiated cell type; adeceleration in the neoplastic progress; or alternatively the slowing ofthe progression of the cancer cells from one stage to the next.

Reduction of growth of cancer cells may be utilized for the treatment ofcancer by the administration, to an individual in need of suchtreatment, of a therapeutically effective amount of the compound of thepresent invention, as described herein.

In a preferred embodiment, the methods of the invention comprise the useof Formula I, wherein at least one of R₆ and R₇ is Br. In furtherpreferred embodiments, the compound is MJDB or MJTB.

The present invention additionally discloses use of a composition ofFormula I, as described above, for preparing a medicament for thetreatment of cancer in mammals.

The term “treatment of cancer” in the context of the present inventionincludes at least one of the following: a decrease in the rate of growthof the cancer (i.e. the cancer still grows but at a slower rate);cessation of growth of the cancerous growth, i.e., stasis of the tumorgrowth, and, in preferred cases, the tumor diminishes or is reduced insize. The term also includes reduction in the number of metastasis,reduction in the number of new metastasis formed, slowing of theprogression of cancer from one stage to the other and a decrease in theangiogenesis induced by the cancer. In most preferred cases, the tumoris totally eliminated. Additionally included in this term is lengtheningof the survival period of the subject undergoing treatment. This termalso encompasses prevention for prophylactic situations or for thoseindividuals who are susceptible to contracting a tumor. Theadministration of the compounds of the present invention will reduce thelikelihood of the individual contracting the disease. In preferredsituations, the individual to whom the compound is administered does notcontract the disease.

The term “cancer” in the context of the present invention includes alltypes of neoplasm whether in the form of solid or non-solid tumors, fromall origins, and includes both malignant and premalignant conditions aswell as their metastasis. In particular this term refers to: carcinoma,sarcoma, adenoma, hepatocellular carcinoma, hepatoblastoma,rhabdomyosarcoma, esophageal carcinoma, thyroid carcinoma,ganglioblastoma, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphagiosarcoma, synovioama, Ewing's tumor, leimyosarcoma,rhabdotheliosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, renal cell carcinoma, hematoma, bile ductcarcinoma, melanoma, choriocarcinoma, seminoma, embryonal carcinoma,Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, smallcell and non-small cell lung carcinoma, bladder carcinoma, epithelialcarcinoma, glioma, astrocyoma, medulloblastoma, craniopharyngioma,ependynoma, pinealoma, retinoblastoma, multiple myeloma, rectalcarcinoma, cancer of the thyroid, head and neck cancer, brain cancer,cancer of the peripheral nervous system, cancer of the central nervoussystem, neuroblastoma, cancer of the endometrium, lymphoproliferativediseases, hematopoietic malignancies including all types of leukemia andlymphoma including: acute myelogenous leukemia, acute myelocyticleukemia, acute lymphocytic leukemia, chronic myelogenous leukemia,chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma,myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, as well asmetastasis of all the above.

More preferably, the cancer is selected from the group consisting ofprostate cancer, breast cancer, skin cancer, colon cancer, lung cancer,pancreatic cancer, lymphoma, leukemia, head and neck cancer, kidneycancer, ovarian cancer, bone cancer, liver cancer or thyroid cancer.Even more preferably, the cancer is selected from leukemia, includinglymphoblastic leukemia, lung carcinoma, melanoma and colon cancer.Additionally, in a preferred embodiment of the method, the compound isadministered at a dosage selected from 1 μg-1000 mg/kg body weight.

In other embodiments of the use of preparing a medicament, themedicament additionally comprises at least one active chemotherapeuticagent other than the compound of Formula I. In certain embodiments, thenovel compound may be administered alongside with traditionalchemotherapeutic drugs that are effective but have considerable sideeffects. The combination of a compound of the invention and thetraditional drug may allow administration of a lesser quantity of thetraditional drug, and thus the side effects experienced by the subjectmay be significantly lower, while a sufficient chemotherapeutic effectis nevertheless achieved.

The present invention additionally discloses a method for the treatmentof cancer in a subject in need thereof, comprising administering to amammal a therapeutically effective amount of a pharmaceuticalcomposition comprising as the active ingredient a compound of thepresent invention, as described above.

There is also provided in the present invention a pharmaceuticalcomposition for the treatment of cancer in mammals, comprising as theactive ingredient a therapeutically effective amount of a compound ofthe invention, as described above,

The invention also provides use of the compounds of the invention in thepreparation of a medicament for reducing the growth of cancer cells, asdescribed herein.

The present invention further discloses a method for preparation of MJDB

or MJTB

comprising:

-   -   i. adding bromine to a solution of methyl jasmonate in CCl₄; and    -   ii. evaporating the CCl₄.

Other jasmonate derivatives of the invention can be made, as describedin the examples below. As further illustrations, an alkyl moiety can beadded at the R₂ position, for example, by using a Grignard reagent atlow temperature.

In addition, a 9,10-epoxide can be obtained from methyl jasmonate usingperoxy acid, This epoxide can react with a compound of the formula RMgXresulting in hydroxyl and alkyl at the 9 and 10 positions (R₆ and R₇,respectively). A substituted alkyl can be added in similar fashion.

Moreover, reacting this epoxide with a compound of the formula ROH orArOH wherein R is alkyl and Ar is aryl under acidic or basic conditionscan result in a hydroxyl and alkyloxy or aryloxy at the 9 and 10positions (R₆ and R₇, respectively). Furthermore, preparing the5,6-enamine of methyl jasmonate will allow for an alkyl at the C₅position (position B). A substituted alkyl can be added in similarfashion.

There is also provided in the present invention a pharmaceuticalcomposition for the treatment of cancer in mammals, comprising as theactive ingredient a therapeutically effective amount of a compound ofthe invention, as described herein, and a pharmaceutically acceptablecarrier.

The term “pharmaceutically acceptable” means approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inanimals and, more particularly, in humans.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic compound is administered. Such pharmaceuticalcarriers can be sterile liquids, such as water and oils, including thoseof petroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like, polyethylene glycols,glycerine, propylene glycol or other synthetic solvents. Water is apreferred carrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene glycol, water, ethanol and thelike.

The composition, if desired, can also contain minor amounts of wettingor emulsifying agents, or pH buffering agents such as acetates, citratesor phosphates. Antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; and agents forthe adjustment of tonicity such as sodium chloride or dextrose are alsoenvisioned.

The compositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides, microcrystallinecellulose, gum tragacanth or gelatin. Oral formulation can includestandard carriers such as pharmaceutical grades of mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate, etc. Examples of suitable pharmaceutical carriers aredescribed in “Remington's Pharmaceutical Sciences” by E. W. Martin. Suchcompositions will contain a therapeutically effective amount of acompound of the invention, preferably in a substantially purified form,together with a suitable amount of carrier so as to provide the form forproper administration to the subject.

The carrier can be selected at times based on the desired form of theformulation. The carrier may also at times have the effect of theimproving the delivery or penetration of the active ingredient to thetarget tissue, for improving the stability of the drug, for slowingclearance rates, for imparting slow release properties, for reducingundesired side effects etc. The carrier may also be a substance thatstabilizes the formulation (e.g. a preservative), for providing theformulation with an edible flavor, etc.

The carriers may be any of those conventionally used and are limitedonly by chemical-physical considerations, such as solubility and lack ofreactivity with the compound of the invention, and by the route ofadministration. The choice of carrier will be determined by theparticular method used to administer the pharmaceutical composition.Accordingly, the carrier may include additives, colorants, diluents,buffering agents, disintegrating agents, moistening agents,preservatives, flavoring agents, and pharmacologically compatiblecarriers. In addition, the carrier may be an adjuvant, which, bydefinition are substances affecting the action of the active ingredientin a predictable way.

Methods of introduction of a pharmaceutical composition comprising acompound of the invention include, but are not limited to, topical,intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous,intranasal, epidural, ophthalmic, and oral routes. The compounds may beadministered by any convenient route, for example by infusion or bolusinjection, by absorption through epithelial linings (e.g., oral mucosa,rectal and intestinal mucosa, etc.), and may be administered togetherwith other therapeutically active agents. It is preferred thatadministration is localized, but it may be systemic. In addition, it maybe desirable to introduce the pharmaceutical compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

It may be desirable to administer the pharmaceutical composition of theinvention locally to the area in need of treatment; this may be achievedby, for example, and not by way of limitation, local infusion duringsurgery topical application, e.g., in conjunction with a wound dressingafter surgery, by injection, by means of a catheter, by means of asuppository, or by means of an implant, said implant being of a porous,non-porous, or gelatinous material. According to some preferredembodiments, administration can be by direct injection e.g., via asyringe, at the site of a tumor or neoplastic or pre-neoplastic tissue.

Pharmaceutical compositions suitable for oral administration may consistof (a) liquid solutions, where an effective amount of the activesubstance is dissolved in diluents, such as water, saline, naturaljuices, alcohols, syrups, etc.; (b) solid dosage forms such as capsules(e.g. the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers), tablets, lozenges(wherein the active substance is flavored, such as with sucrose andacacia or tragacanth, or the active substance is in an inert base, suchas gelatin and glycerin), and troches, each containing a predeterminedamount of the active ingredient as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; (e) suitable emulsions; (f)liposome formulation; and others.

In yet another embodiment, the composition is prepared for topicaladministration, e.g. as an ointment, a gel a drop or a cream. Fortopical administration to body surfaces using, for example, creams,gels, drops, ointments and the like, the compounds of the presentinvention can be prepared and applied in a physiologically acceptablediluent with or without a pharmaceutical carrier. The present inventionmay be used topically or transdermally to treat cancer, for example,melanoma. Adjuvants for topical or gel base forms may include, forexample, sodium carboxymethylcellulose, polyacrylates,polyoxyethylene-polyoxypropylene-block polymers, polyethylene glycol andwood wax alcohols.

For directed internal topical applications, the pharmaceuticalcomposition may be in the form of tablets or capsules, which can containany of the following ingredients, or compounds of a similar nature: abinder such as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose; a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; or a glidant such as colloidal silicon dioxide.When the dosage unit form is a capsule, it can contain, in addition tomaterials of the above type, a liquid carrier such as a fatty oil. Inaddition, dosage unit forms can contain various other materials whichmodify the physical form of the dosage unit, for example, coatings ofsugar, shellac, or other enteric agents.

A compound of the present invention can be delivered in a controlledrelease system. In one embodiment, an infusion pump may be used toadminister a compound of the invention, such as one that is used fordelivering chemotherapy to specific organs or tumors (see Buchwald etal., 1980, Surgery 88: 507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In a preferred form, a compound of the invention is administeredin combination with a biodegradable, biocompatible polymeric implant,which releases the compound over a controlled period of time at aselected site. Examples of preferred polymeric materials includepolyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid,polyethylene vinyl acetate, copolymers and blends thereof (See, Medicalapplications of controlled release, Langer and Wise (eds.), 1974, CRCPres., Boca Raton, Fla.). In yet another embodiment, a controlledrelease system can be placed in proximity of the therapeutic target,thus requiring only a fraction of the systemic dose.

At times, the active compound may be made into aerosol formulations tobe administered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer.

Furthermore, at times, the pharmaceutical compositions may be formulatedfor parenteral administration (subcutaneous, intravenous, intraarterial,or intramuscular injection) and may include aqueous and non-aqueous,isotonic sterile injection solutions, which can contain anti-oxidants,buffers, bacteriostats, and solutes that render the formulation isotonicwith the blood of the intended recipient, and aqueous and non-aqueoussterile suspensions that include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. Oils such aspetroleum, animal, vegetable, or synthetic oils and soaps such as fattyalkali metal, ammonium, and triethanolamine salts, and suitabledetergents may also be used for parenteral administration. The aboveformulations may also be used for direct intra-tumoral injection.Further, in order to minimize or eliminate irritation at the site ofinjection, the compositions may contain one or more nonionicsurfactants. Suitable surfactants include polyethylene sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol.

The parenteral formulations can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, water, for injections, immediatelyprior to use. Extemporaneous injection solutions and suspensions can beprepared from sterile powders, granules, and tablets of the kindpreviously described and known in the art.

The amount of a compound of the invention that will be effective in thetreatment of a particular disorder or condition, including cancer, willdepend on the nature of the disorder or condition, and can be determinedby standard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease or disorder,and should be decided according to the judgment of the practitioner andeach patient's circumstances. A preferred dosage will be within therange of 0.01-1000 mg/kg of body weight, more preferably, 0.1 mg/kg to100 mg/kg and even more preferably 1 mg/kg to 10 mg/kg. Effective dosesmay be extrapolated from dose-response curves derived from in vitro oranimal model test bioassays or systems.

A “therapeutic” treatment is a treatment administered to a subject whoexhibits signs of pathology for the purpose of diminishing oreliminating those signs A “therapeutically effective amount” of acompound of the invention is that amount of compound which is sufficientto provide a beneficial effect to the subject to which the compound isadministered.

Patients in need thereof may suffer from a disease such as cancer or mayhave been determined to have a greater susceptibility to such disease.Thus, the method of treatment according to the present inventionincludes both therapeutic and prophylactic utility.

A compound of the invention can be tested in vivo for the desiredtherapeutic or prophylactic activity as well as for determination of atherapeutically effective dosage. For example, such compounds can betested in suitable animal model systems prior to testing in humans,including, but not limited to, rats, mice, chicken, cows, monkeys,rabbits, and the like. For in vivo testing, prior to administration tohumans, any animal model system known in the art may be used.

When the above-described compounds include one or more chiral centers,the stereochemistry of such chiral centers can independently be in the Ror S configuration, or a mixture of the two. The chiral centers can befurther designated as R or S or R,S or d,D, l,L or d,l, D,L.

The term “C₁ to C₁₂ alkyl” denotes such radicals as methyl, ethyl,n-propyl, isopropyl, n-butyl, iso-butyl, sec-butyl, tert-butyl, amyl,tert-amyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and thelike. Preferred “C₁ to C₁₂ alkyl” groups are methyl, ethyl, iso-butyl,sec-butyl and iso-propyl. Similarly, the term “C₁ to C₁₂ alkylene”denotes radicals of 1 to 12 carbons connected to two other parts in thecompound.

The term “C₁ to C₁₂ substituted alkyl denotes groups that aresubstituted by one or more, and preferably one or two substituentsselected from halogen, hydroxy, protected hydroxy, oxo, protected oxo,C₃ to C₇ cycloalkyl, phenyl, substituted phenyl, naphthyl, amino,protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, guanidino, protectedguanidino, heterocyclic ring, substituted heterocyclic ring, imidazolyl,indolyl, pyrrolidinyl, C₁ to C₁₂ alkoxy. C₁ to C₁₂ acyl, C₁ to C₁₂acyloxy, nitro, carboxy, protected carboxy, carbamoyl, carboxamide,protected carboxamide, N—(C₁ to C₁₂ alkyl)carboxamide, protected N—(C₁to C₁₂ alkyl)carboxamide. N,N-di(C₁ to C₁₂ alkyl)carboxamide, cyano,methylsulfonylamino, thiol, C₁ to C₁₀ alkylthio or C₁ to C₁₀alkylsulfonyl groups. The substituted alkyl groups may be substitutedonce or more, and preferably once or twice, with the same or withdifferent substituents. A preferred substitution is halo.

The term “protected oxo” denotes a carbon atom bonded to two alkoxygroups thereby forming an acyclic or cyclic acetal or ketal moiety. Theterm “C₁ to C₁₂ alkoxy” as used herein denotes groups such as methoxy,ethoxy, n-propoxy, isopropoxy, n-butoxy, t-butoxy and like groups. Apreferred alkoxy is methoxy. The term “C₁ to C₁₂ substituted alkoxy”means the alkyl portion of the alkoxy can be substituted in the samemanner as in relation to C₁ to C₁₂ substituted allyl. A preferredsubstitution is halo. Similarly, the term “C₁ to C₁₂ phenylalkoxy” asused herein means “C₁ to C₁₂ alkoxy” bonded to a phenyl radical.

The term “C₁ to C₁₂ acyloxy” denotes herein groups such as formyloxy,acetoxy, propionyloxy, butyryloxy, pivaloyloxy, pentanoyloxy;hexanoyloxy, heptanoyloxy, octanoyloxy, nonanoyloxy, decanoyloxy,undecanoyloxy, dodecanoyloxy and the like.

Similarly, the term “C₁ to C₁₂ acyl” encompasses groups such as formyl,acetyl, propionyl, butyryl, pentanoyl, pivaloyl, hexanoyl, heptanoyl,octanoyl, nonanoyl, decanoyl, undecanoyl, dodecanoyl, benzoyl and thelike. Preferred acyl groups are acetyl and benzoyl.

The term “C₁ to C₁₂ substituted acyl” denotes the acyl group substitutedby one or more, and preferably one or two, of the substituents definedabove for alkyl. The substituent term “C₃ to C₇ cycloalkyl” includes thecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl rings.Similarly, a substituent that can be C₃ to C₇ cycloalkyl” can also be“C₅ to C₇ cycloalkyl,” which includes the cyclopentyl, cyclohexyl orcycloheptyl rings.

The substituent term “C₃ to C₇ substituted cycloalkyl” or “C₅ to C₇substituted cycloalkyl” indicates the above cycloalkyl rings substitutedby one or two of the substituents defined above for alkyl. The term“cycloalkylene” means a cycloalkyl, as defined above, where thecycloalkyl radical is bonded at two positions connecting together twoseparate additional groups. Similarly, the term “substitutedcycloalkylene” means a cycloalkylene where the cycloalkyl radical isbonded at two positions connecting together two separate additionalgroups and further bearing at least one additional substituent.

Similarly, the term “substituted C₅ to C₇ cycloalkenylene” means acycloalkenylene further substituted by halogen, hydroxy, protectedhydroxy, C₁ to C₁₀ alkylthio, C₁ to C₁₀ alkylsulfoxide, C₁ to C₁₀alkylsulfonyl, C₁ to C₁₀ substituted alkylthio, C₁ to C₁₀ substitutedalkylsulfoxide, C₁ to C₁₀ substituted alkylsulfonyl, C₁ to C₁₂ alkyl, C₁to C₁₂ alkoxy, C₁ to C₁₂ substituted alkyl, C₁ to C₁₂ alkoxy, oxo,protected oxo, (monosubstituted)amino, (disubstituted)amino,trifluoromethyl, carboxy, protected carboxy, phenyl, substituted phenyl,phenylthio, phenylsulfoxide, phenylsulfonyl, amino, or protected aminogroup.

The term “heterocycle” or “heterocyclic ring” denotes optionallysubstituted five-membered to eight-membered rings that have 1 to 4heteroatoms, such as oxygen, sulfur and/or nitrogen, in particularnitrogen, either alone or in conjunction with sulfur or oxygen ringatoms. These five-membered to eight-membered rings may be saturated,fully unsaturated or partially unsaturated, with fully saturated ringsbeing preferred. Preferred heterocyclic rings include morpholino,piperidinyl, piperazinyl, 2-amino-imidazoyl, tetrahydrofurano, pyrrolo,tetrahydrothiophenyl, hexylmethyleneimino and heptylmethyleneimino.

The term “substituted heterocycle” or “substituted heterocyclic ring”means the above-described heterocyclic ring is substituted with, forexample, one or more, and preferably one or two, of the substituentsdefined above for alkyl. The term “heteroaryl” means a heterocyclicaromatic derivative which is a five-membered or six-membered ring systemhaving from 1 to 4 heteroatoms, such as oxygen, sulfur and/or nitrogen,in particular nitrogen, either alone or in conjunction with sulfur oroxygen ring atoms. Examples of heteroaryls include pyridinyl,pyrimidinyl, and pyrazinyl pyridazinyl, pyrrolo, furano, oxazolo,isoxazolo, phthalimido, thiazolo and the like;

The term “substituted heteroaryl” means the above-described heteroarylis substituted with, for example, one or more, and preferably one ortwo, substituents which are the same or different, of the substituentsdefined above for alkyl. The term “substituted phenyl” specifies aphenyl group substituted with one or more, and preferably one or to,moieties chosen from the substituents defined above for alkyl.

The term “phenoxy” denotes a phenyl bonded to an oxygen atom, whereinthe binding to the rest of the molecule is through the oxygen atom. Theterm “substituted phenoxy” specifies a phenoxy group substituted withone or more, and preferably one or two, moieties chosen from thesubstituents defined above for alkyl.

The term “aryl” refers to an aromatic group having at least onecarbocyclic aromatic group or heterocyclic aromatic group, which may beunsubstituted or substituted by one or more groups selected fromhalogen, haloalkyl, hydroxy, alkoxy carbonyl, amido, alkylamido,dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxy or thio orthioalkyl. Nonlimiting examples of aryl rings are phenyl, naphthyl,pyranyl, pyrrolyl, pyrazinyl, pyrimidinyl, pyrazolyl, pyridinyl,furanyl, thiophenyl, thiazolyl, imidazolyl, isoxazolyl, and the like.The term “aryloxy” refers to an “aryl” group bonded to an oxygen atom,wherein the binding to the rest of the molecule is through the oxygenatom.

The terms “halo” and “halogen” refer to the fluoro, chloro, bromo oriodo atoms. There can be one or more halogens, which are the same ordifferent.

The term “(monosubstituted) amino” refers to an amino group with onesubstituent chosen from the group consisting of phenyl, substitutedphenyl, C₁ to C₁₂ alkyl, C₁ to C₁₂ substituted alkyl C₁ to C₁₂ acyl, C₁to C₁₂ substituted acyl, C₂ to C₁₂ alkenyl, C₂ to C₁₂ substitutedalkenyl, C₂ to C₁₂ alkynyl, C₂ to C₁₂ substituted alkynyl, C₇ to C₁₈phenylalkyl, C₇ to C₁₈ substituted phenylalkyl, heterocyclic ring,substituted heterocyclic ring, C₁ to C₁₂ heterocycloalkyl and C₁ to C₁₂substituted heterocycloalkyl. The (monosubstituted)amino canadditionally have an amino-protecting group as encompassed by the term“protected (monosubstituted)amino”.

The term “(disubstituted)amino” refers to an amino group with twosubstituents chosen from the group consisting of phenyl, substitutedphenyl, C₁ to C₁₂ allyl, C₁ to C₁₂ substituted alkyl, C₁ to C₁₂ acyl, C₂to C₁₂ alkenyl, C₂ to C₁₂ alkynyl, C₇ to C₁₅ phenylalkyl, C₇ to C₁₈substituted phenylalkyl, C₁ to C₁₂ heterocycloalkyl and C₁ to C₁₂substituted heterocycloalkyl. The two substituents can be the same ordifferent.

The term “amino-protecting group” as used herein refers to substituentsof the amino group commonly employed to block or protect the aminofunctionality while reacting other functional groups of the molecule.The term “protected (monosubstituted)amino” means there is anamino-protecting group on the monosubstituted amino nitrogen atom. Inaddition, the term “protected carboxamide” means there is anamino-protecting group on the carboxamide nitrogen. Similarly, the term“protected N—(C₁ to C₁₂ alkyl)carboxamide” means there is anamino-protecting group on the carboxamide nitrogen.

The species of amino-protecting group employed is not critical so longas the derivatized amino group is stable to the conditions of thesubsequent reaction(s) and can be removed at the appropriate pointwithout disrupting the remainder of the compounds. Preferredamino-protecting groups are Boc, Cbz and Fmoc. Further examples ofamino-protecting groups embraced by the above term are well known inorganic synthesis and the peptide art and are described by, for example,T. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” 2nd ed., John Wiley and Sons, New York. N.Y., 1991, Chapter7, M. Bodanzsky, “Principles of Peptide Synthesis,” 1st and 2nd reviseded., Springer-Verlag, New York, N.Y., 1984 and 1993, and Stewart andYoung, “Solid Phase Peptide Synthesis,” 2nd ed., Pierce Chemical Co.,Rock-ford, Ill. 1984, each of which is incorporated herein by reference.The related term “Protected amino” defines an amino group substitutedwith an amino-protecting group discussed above.

The term “protected guanidino” as used herein refers to an“amino-protecting group”on one or two of the guanidino nitrogen atoms,Examples of “protected guanidino” groups are described by T. W. Greeneand P. G. M. Wuts; M. Bodanzsky; and Stewart ad Young, supra.

The term “thio” refers to —SH or, if between two other groups, —S—. Theterm “C₁ to C₁₀ alkylene thio” refers to a one to ten carbon alkylenechain with a thio at any point along the chain. The term “C₁ to C₁₀substituted alkylene thio” refers to a C₁ to C₁₀ alkylene thio groupthat is substituted at one or more of the alkylene positions (in thesame way as “substituted alkylene,” as described above).

The term “sulfonyl” refers to —S(O)₂—. The term “C₁ to C₁₀ alkylenesulfonyl” refers to a one to ten carbon alkylene chain with a sulfonylat any point along the chain. The term “C₁ to C₁₀ substituted alkylenesulfonyl” refers to a C₁ to C₁₀ alkylene sulfonyl group that issubstituted at one or more of the alkylene positions (in the same way as“substituted allylene,” as described above).

The term “sulfinyl” refers to —S(O)—. The term “C₁ to C₁₀ allylenesulfinyl” refers to a one to ten carbon alkylene chain with a sulfinylat any point along the chain. The term “C₁ to C₁₀ substituted alkylenesulfinyl” refers to a C₁ to C₁₀ alkylene sulfinyl group that issubstituted at one or more of the alkylene positions (in the same way as“substituted alkylene,” as described above).

The term “oxy” refers to —O—. The terms “C₁ to C₁₀ allylene oxy,” “C₁ toC₁₀ alkylene dioxy” and “C₁ to C₁₀ alkylene trioxy” refer to a one toten carbon alkylene chain with, respectively, one, two or three —O— atany point along the chain, provided that no two oxygen atoms areconsecutive, and provided that any two oxygen atoms are separated by atleast two carbons. The terms “C₁ to C₁₀ substituted alkylene oxy,” “C₁to C₁₀ substituted allylene dioxy” and “C₁ to C₁₀ substituted alkylenetrioxy” refer, respectfully to “C₁ to C₁₀ alkylene oxy,” “C₁ to C₁₀alkylene dioxy” and “C₁ to C₁₀ alkylene trioxy” that are substituted atone or more of the alkylene positions (in the same way as “substitutedalkylene,” as described above).

The term “carboxy-protecting group” as used herein refers to one of theester derivatives of the carboxylic acid group commonly employed toblock or protect the carboxylic acid group while reactions are carriedout on other functional groups on the compound. Examples of these groupsare found in E. Haslam, “Protective Groups in Organic Chemistry.” J. G.W. McOmie, Ed., Plenum Press, New York N.Y., 1973, Chapter 5, and T. A.Greene and P. G. M. Wuts, “Protective Groups in Organic Synthesis,” 2ndPd, John Wiley and Sons, New York, N.Y., 1991, Chapter 5, each of whichis incorporated herein by reference. A related term is “protectedcarboxy,” which refers to a carboxy group substituted with one of theabove carboxy-protecting groups.

The term “hydroxy-protecting group” refers to readily cleavable groups)bonded to hydroxyl groups. The species of hydroxy-protecting groups isnot critical so long as the derivatized hydroxyl group is stable to theconditions of subsequent reaction(s) and can be removed at theappropriate point without disrupting the remainder of the molecule.Examples of hydroxy-protecting groups are described by C. B. Reese andE. Haslam “Protective Groups in Organic Chemistry,” J. G. W. McOmie. Ed.Plenum Press. New York, N.Y., 1973, Chapters 3 and 4, respectively, andT. W. Greene and P. G. M. Wuts, “Protective Groups in OrganicSynthesis,” 2nd ed., John Wiley and Sons, New York, N.Y., 1991, Chapters2 and 3. Related terms are “protected hydroxy,” and “protectedhydroxymethyl” which refer to a hydroxy or hydroxymethyl substitutedwith one of the above hydroxy-protecting groups.

The term “C₁ to C₁₀ alkylthio” refers to sulfide groups such asmethylthio, ethylthio, n-propylthio, isopropylthio, n-butylthio,t-butylthio and like groups.

The term “C₁ to C₁₀ alklylsulfoxide” indicates sulfoxide groups such asmethylsulfoxide, ethylsulfoxide, n-propylsulfoxide, isopropylsulfoxide,n-butylsulfoxide, sec-butylsulfoxide and the like. The term “C₁ to C₁₀alkylsulfonyl” encompasses groups such as methylsulfonyl, ethylsulfonyl,n-propylsulfonyl, isopropylsulfonyl, n-butylsulfonyl, t-butylsulfonyland the like. it should also be understood that the above thio,sulfoxide or sulfonyl groups can be at any point on the alkyl chain(e.g., 2-methylmercaptoethyl).

The terms “C₁ to C₁₀ substituted alkylthio,” “C₁ to C₁₀ substitutedalkylsulfoxide,” and “C₁ to C₁₀ substituted alkylsulfonyl.” denote theC₁ to C₁₀ alkyl portion of these groups may be substituted as describedabove in relation to “substituted alkyl.”

The terms “phenylthio,” “phenylsulfoxide,” and “phenylsulfonyl” specifya thiol, a sulfoxide, or sulfone, respectively, containing a phenylgroup. The terms “substituted phenylthio,” “substitutedphenylsulfoxide,” and “substituted phenylsulfonyl” means that the phenylof these groups can be substituted as described above in relation to“substituted phenyl.”

The term “C₁ to C₁₂ alkylaminocarbonyl” means a C₁ to C₁₂ alkyl attachedto a nitrogen of the aminocarbonyl group. Examples of C₁ to C₁₂alkylaminocarbonyl include methylaminocarbonyl, ethylaminocarbonyl,propylaminocarbonyl and butylaminocarbonyl. The term “C₁ to C₁₂substituted alklylaminocarbonyl” denotes a substituted alkyl bonded to anitrogen of the aminocarbonyl group, which alkyl may be substituted asdescribed above in relation to C₁ to C₁₂ substituted alkyl.

The term “C₁ to C₁₂ alkoxycarbonyl” means a “C₁ to C₁₂ alkoxy” groupattached to a carbonyl group. The term “C₁ to C₁₂ substitutedalkoxycarbonyl” denotes a substituted alkoxy bonded to the carbonylgroup, which alkoxy may be substituted as described above in relation to“C₁ to C₁₂ substituted alkyl.”

The term “phenylaminocarbonyl” means a phenyl attached to a nitrogen ofthe aminocarbonyl group. The term “substituted phenylaminocarbonyl”denotes a substituted phenyl bonded to a nitrogen of the aminocarbonylgroup, which phenyl may be substituted as described above in relation tosubstituted phenyl.

The term “C₁ to C₁₂ alkylaminothiocarbonyl” means a C₁ to C₁₂ alkylattached to an aminothiocarbonyl group, wherein the alkyl has the samemeaning as defined above.

The term “C₁ to C₁₂ substituted alkylaminothiocarbonyl” denotes asubstituted alkyl bonded to an aminothiocarbonyl group, wherein thealkyl may be substituted as described above in relation to C₁ to C₁₂substituted alkyl.

The term “phenylaminothiocarbonyl” means a phenyl attached to anaminothiocarbonyl group, wherein the phenyl has the same meaning asdefined above.

The term “substituted phenylaminothiocarbonyl” denotes a substitutedphenyl bonded to an aminothiocarbonyl group, wherein phenyl may besubstituted as described above in relation, to substituted phenyl.

The term “substituted C₁ to C₁₂ alkylene” means a C₁ to C₁₂ alkyl groupwhere the alkyl radical is bonded at two positions connecting togethertwo separate additional groups and further bearing an additionalsubstituent.

The terms “cyclic C₂ to C₇ alkylene,” “substituted cyclic C₂ to C₇alkylene,” “cyclic C₂ to C₇ heteroalkylene,” and “substituted cyclic C₂to C₇ heteroalkylene,” defines such a cyclic group bonded (“fused”) tothe phenyl radical resulting in a bicyclic ring system. The cyclic groupmay be saturated or contain one or two double bonds. Furthermore, thecyclic group may have one or two methylene or methine groups replaced byone or two oxygen, nitrogen or sulfur atoms that are the cyclic C₂ to C₇heteroalkylene.

The cyclic alkylene or heteroalkylene group may be substituted once ortwice by the same or different substituents which, if appropriate, canbe connected to another part of the compound (e.g., alkylene) selectedfrom the group consisting of the following moieties: hydroxy, protectedhydroxy, carboxy, protected carboxy, oxo, protected oxo, C₁ to C₄acyloxy, formyl, C₁ to C₁₂ acyl, C₁ to C₁₂ alkyl C₁ to C₇ alkoxy, C₁ toC₁₀ alkylthio. C₁ to C₁₀ alkylsulfoxide C₁ to C₁₀ alkylsulfonyl, halo,amino, protected amino, (monosubstituted)amino, protected(monosubstituted)amino, (disubstituted)amino, hydroxymethyl or aprotected hydroxymethyl.

The cyclic alkylene or heteroalkylene group fused onto the benzeneradical can contain two to ten ring members, but it preferably containsthree to six members. Examples of such saturated cyclic groups are whenthe resultant bicyclic ring system is 2,3-dihydro-indanyl and a tetralinring. When the cyclic groups are unsaturated, examples occur when theresultant bicyclic ring system is a naphthyl ring or indolyl. Examplesof fused cyclic groups which each contain one nitrogen atom and one ormore double bond, preferably one or two double bonds, are when thebenzene radical is fused to a pyridino, pyrano, pyrrolo, pyridinyl,dihydropyrrolo, or dihydropyridinyl ring. Examples of fused cyclicgroups which each contain one oxygen atom and one or two double bondsare when the benzene radical ring is fused to a furo, pyrano,dihydrofurano, or dihydropyrano ring. Examples of fused cyclic groupswhich each have one sulfur atom and contain one or two double bonds arewhen the benzene radical is fused to a thieno, thiopyrano, dihydrothienoor dihydrothiopyrano ring. Examples of cyclic groups that contain twoheteroatoms selected from sulfur and nitrogen and one or two doublebonds are when the benzene radical ring is fused to a thiazolo,isothiazolo, dihydrothiazolo or dihydroisothiazolo ring. Examples ofcyclic groups which contain two heteroatoms selected from oxygen andnitrogen and one or two double bonds are when the benzene ring is fusedto an oxazolo, isoxazolo, dihydrooxazolo or dihydroisoxazolo ring.Examples of cyclic groups which contain two nitrogen heteroatoms and oneor two double bonds occur when the benzene ring is fused to a pyrazolo,imidazolo, dihydropyrazolo or dihydroimidazolo ring or pyrazinyl.

The term “carbamoyl” means an —NC(O)— group where the radical is bondedat two positions connecting two separate additional groups.

One or more of the compounds of the invention, may be present as a salt.The term “salt” encompasses those salts that form with the carboxylateanions and amine nitrogens and include salts formed with the organic andinorganic anions and cations discussed below. Furthermore, the termincludes salts that form by standard acid-base reactions with basicgroups (such as amino groups) and organic or inorganic acids. Such acidsinclude hydrochloric, hydrofluoric, trifluoroacetic, sulfuric,phosphoric, acetic, succinic, citric, lactic, maleic, fumaric, palmitic,cholic, pamoic, mucic, D-glutamic, D-camphoric, glutaric, phthalic,tartaric, lauric, stearic, salicyclic, methanesulfonic, benzenesulfonic,sorbic, picric, benzoic, cinnamic, and like acids.

The term “organic or inorganic cation” refers to counter-ions for thecarboxylate anion of a carboxylate salt. The counter-ions are chosenfrom the alkali and alkaline earth metals, (such as lithium, sodium,potassium, barium, aluminum and calcium); ammonium and mono-, di- andtri-alkyl amines such as trimethylamine, cyclohexylamine; and theorganic cations, such as dibenzylammonium, benzylammonium,2-hydroxyethylammonium, bis(2-hydroxyethyl)ammonium,phenylethylbenzylammonium, dibenzylethylenediammonium, and like cations.See, for example, “Pharmaceutical Salts,” Berge et al., J. Pharm. Sci.,66:1-19 (1977), which is incorporated herein by reference. Other cationsencompassed by the above term include the protonated form of procaine,quinine and N-methylglucosamine, and the protonated forms of basic aminoacids such as glycine, ornithine, histidine, phenylglycine, lysine andarginine. Furthermore, any zwitterionic form of the instant compoundsformed by a carboxylic acid and an amino group is referred to by thisterm. For example, a cation for a carboxylate anion will exist when aposition is substituted with a (quaternary ammonium)methyl group. Apreferred cation for the carboxylate anion is the sodium cation.

The compounds of the invention can also exist as solvates and hydrates.Thus, these compounds may crystallize with, for example, waters ofhydration, or one, a number of, or any fraction thereof of molecules ofthe mother liquor solvent. The solvates and hydrates of such compoundsare included within the scope of this invention.

One or more compounds of the invention, even when in a library, can bein the biologically active ester form, such as the non-toxic,metabolically-labile ester-form. Such ester forms induce increased bloodlevels and prolong the efficacy of the corresponding non-esterifiedforms of the compounds. Ester groups which can be used include the loweralkoxymethyl groups, for example, methoxymethyl, ethoxymethyl,isopropoxymethyl and the like; the —(C₁ to C₁₂) alkoxyethyl groups, forexample methoxyethyl, ethoxyethyl, propoxyethyl, isopropoxyethyl and thelike; the 2-oxo-1,3-diooxlen-4-ylmethyl groups, such as5-methyl-2-oxo-1,3-dioxolen-4-ylmethyl,5-phenyl-2-oxo-1,3-dioxolen-4-ylmethyl and the like: the C₁ to C₁₀alkylthiomethyl groups, for example methylthiomethyl, ethylthiomethyl,iso-propylthiomethyl and the like; the acyloxymethyl groups, for examplepivaloyloxymethyl, pivaloyloxyethyl, -acetoxymethyl and the like; theethoxycarbonyl-1-methyl group; the -acetoxyethyl; the 1-(C₁ to C₁₂alkyloxycarbonyloxy)ethyl groups such as the 1-(ethoxycarbonyloxy)ethylgroup; and the 1-(C₁ to C₁₂ allklaminocarbonyloxy)ethyl groups such asthe 1-(methylaminocarbonyloxy)ethyl group.

It should be understood that any position of the claimed invention hasup to three serial “substitutions.” For example, a “substituted alkyl”that is substituted with a “substituted phenyl” that is, in turn,substituted with a “substituted alkyl” can, in turn, be substituted byone more group and no longer further substituted. However, it shouldalso be understood that the invention contemplates, if appropriate, morethan three parallel substitutions. For example, if appropriate, morethan three hydrogens on an alkyl moiety may be substituted with any oneor more of a variety of groups, including halo and hydroxy.

EXAMPLES Experimental Procedures

Synthesis of MJDB

A solution of methyl jasmonate in CCl₄ at −20° C. was treated withbromine until a yellow color was kept for 5 minutes. The solvent wasthen evaporated and the yellowish residue chromatographed on an MeOHwashed Silica gel column (VLC) and eluted with hexane/5-10% EtOAc.

A 1:1 mixture of two possible racemates was obtained.

Mass spectra: m/z 384 (Br₂), Rf=0.8 on silica gel eluted withhexane/EtOAc 1:1. C NMR (CDCl3): (C-1 to C-13): 172.11/172.3; 37.1/373;38.0/38; 29.5/29.6; 38.8/39.1; 218.4/219.1; 51.7 (for both); 27.0/27.2;57.8/55.8; 60.7/60.2; 35.7/36.1; 12.4 (for both). 51.1/51.3 ppm. H NMR(CDCl3): 2.39-2.41 (H-2 and 3); 1.94-2.14 (H-4, H-7 and H-11); 2.74-2.75(H-5); 1.61 (H-8) 4.62 and 4.89 (H-9); 4.14 (H-10) 1.12 (H-12); 3.75(OMe) ppm.

Synthesis of MJTB

MJ in CCl4 was treated over night with 10 equivalents of bromine. Thesolvent and excess of bromine were evaporated under vacuum.Recrystallizations from iPrOH gave one tetra bromo isomer andrecrystallizations from EtOH gave a second isomer. The compounds wereidentified by NMR, MS and one isomer by an X-ray diffraction analysis.

Synthesis Of MJS72f5

To a stirred solution of (±)-MJ (111 mg, 0.49 mmol) in methanol (5 mL)at 0° C. was added dropwise a solution of I₂ (580 mg. 2.28 mmol) inmethanol (15 mL). The mixture was stirred for 0.5 hr at 0° C. in thedark allowed to warm up to room temperature and then stirred further for48 hr. The solvent was then evaporated, saturated aq. Na₂SO₃ (10 mL) wasadded to the residue, and extracted with ether (2×10 mL). The combinedorganic layer was dried over MgSO₄ and concentrated in vacuo. Theresidue was purified by VLC (EtOAc/petroleum ether 1:4) affording theproduct (42 mg, 22%) as a colorless oil.

Synthesis of MJS99f7

Fluorine, at a concentration of 3% in N₂, was passed as a slow streamthrough a cold (−75° C.) and vigorously stirred solution of thesubstrate(±)-MJ (2.1 gr, 9.37 mmol) dissolved in 250 mL of CFCl₃, 200 mLof CHCl₃, and 50 mL of ethanol. The reaction was carried out for 3 hr.then the mixture was poured into 500 mL of water, washed with saturatedaq. NaHCO₃ and extracted with CH₂Cl₂. The combined organic layer wasdried over MgSO₄ and concentrated in vacuo. The residue was purified byVLC (EtOAc/petroleum ether 1:9) affording a mixture of diastereomers(950 mg, 39%) as a red oil.

Synthesis of MJS85f4

To a stirred solution of (±)-MJ (246 mg, 1.10 mmol) in dry THF (6 mL) at0° C. was added dropwise a solution of I₂ (1.17 gr, 4.6 mmol) in THF (15mL). The mixture was stirred for 3 hr at 0° C. in the dark, allowed towarm up to room temperature and then stirred further for 48 hr. Aftercompletion, saturated aq. Na₂SO₃ (10 mL) was added to the residue, andextracted with ether (2×10 mL). The combined organic layer was driedover MgSO₄ and concentrated in vacuo. The residue was purified by VLC(EtOAc/petroleum ether 1:4) affording iodination products.

Synthesis OF MJS81f3

To a stirred solution of (±)-MJ (78 mg, 0.349 mmol) in CCl₄ (5 mL) at−10° C. was bubbled Cl₂ that was made in situ from concentrated HCl (5mL) and KMnO₄ (800 mg). The mixture was stirred at −10° C. for 2 hr. Thesolvent was then evaporated and the residue purified by VLC(EtOAc/petroleum ether 1:19) affording chlorination products.

Cytotoxicity Assay Used in Examples

Measurement of reduction in the number of living cells was determinedby, the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay(Promega, Madison Wis.). Upon completion of a given experiment, MTS (atetrazolium compound) at 333 μg/ml+phenazine methosulfate (at 25 μM) wasadded to each well of the 96-well plate for 1 hour at 37° C. Thisallowed for development of a color reaction in which dehydrogenasesreduce the MTS in metabolically active cells. Since the cells were notwashed before the addition of MTS, there were no potentially looselyadherent or non-adherent cells that could have been problematic. SolubleMTS formazan product was measured at a wavelength of 490 nm using aCERES 900 HDI ELISA reader (Bio-Tek Instruments, Inc, Highland Park,Vt.), Optical density is directly proportional to the number of livingcells in culture. Cytotoxicity (%) was calculated in the following way:[(OD of control cells−OD of drug-treated cells)/OD of controlcells]×100.

Example 1 MJDB is Highly Cytotoxic Towards Leukemia Cells, and Non-toxicTowards Healthy Lymphocytes

In order to test the toxicity of MJDB towards human leukemia cells,peripheral blood lymphocytes from chronic lymphocytic leukemia (CLL)patients were harvested. These cells were shown to contain practically100% cancer cells, as determined by flow cytometric analysis of the CD5and CD19 markers upon the cell surface. Peripheral blood lymphocytesfrom healthy donors were similarly harvested. Cells were seeded at1.5×10⁴/well, in 96-well plates and MJDB was added for 1 day at severalconcentrations indicated in FIG. 1. The optical density that representedviable cells was determined by the CellTiter 96 Aqueous Non-RadioactiveCell Proliferation Assay (Promega, Madison, Wis.); an assay in whichviable cells produce a colored product, as described above. This assayis quantitative, as the amount of color produced is read using an ELISAreader. Cytotoxicity was calculated as the % of control untreatedcultures, mean±SD; n=3.

The cytotoxicity of MJDB towards peripheral blood lymphocytes from CLLpatients was plotted versus its cytotoxicity towards peripheral bloodlymphocytes from healthy donors (represented by diamonds). See FIG. 1.MJDB was clearly and significantly (P<0.05) more cytotoxic towardsperipheral blood lymphocytes from chronic lymphocytic leukemia (CLL)patients than towards peripheral blood lymphocytes from healthy donors.MJDB is highly and selectively cytotoxic towards cancer cells from CLLpatients, while cytotoxicity is minimal towards lymphocytes from healthydonors.

Example 2 MJDB is Far More Cytotoxic than Previously Studied Jasmonates,as Shown Against Four Diverse Human Malignant Cell Lines

The cytotoxicity of MJDB was compared to that of the previously studiedjasmonate, methyl jasmonate (MJ), which was the most effective jasmonatedisclosed in U.S. Pat. No. 6,469,061. The cytotoxicity of thesecompounds was compared as seen when each was applied to four humanmalignant cell lines originating in lymphoblastic leukemia, lungcarcinoma melanoma, or colon carcinoma.

Molt-4 lymphoblastic leukemia cells (at 1.5×10⁴/well), 3LL lungcarcinoma cells (at 4×10³/well), B16 melanoma cells (at 4×10³/well), orHCT116 colon carcinoma cells (at 4×10³/well) were seeded in 96-wellplates and methyl jasmonate (MJ) or MJDB at 0.5 mM were added for 1 day.Optical density representing viable cells was determined by theCellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (describedabove). Cytotoxicity was calculated as the % of control untreatedcultures, mean±SD. n=3.

The percentage of cytotoxicity of methyl jasmonate (MJ, white columns)or MJDB (filled columns) is illustrated. FIG. 2 clearly indicates thatthe newly synthesized compound MJDB is highly superior in its cytotoxiceffect upon the various cancer cell lines, as compared to the previouslystudied methyl jasmonate. (Statistical studies showed P<0.05 for theseresults).

Example 3 MJDB is Effective Against Either Cells Expressing Wild Type orMutant p53

The effect of MJDB towards cells expressing a mutated form of thepro-apoptotic tumor suppressor gene, p53, was shown. Aberrant p53expression occurs in about 50% of human cancers and contributes to drugresistance and the ensuing failure of chemotherapy and irradiation incancer patients. Consequently, the ability to kill mutant p53-expressingcells is of high clinical significance.

A system of two B lymphoma clones originating from the same cell line(29M4.1) was used. These clones differed solely in the expression ofwild type versus mutant p53. More specifically, 29M4.1 cells were seededat 2.5×10⁴/well, in 96-well plates and MJDB were added for 1 day atseveral concentrations. The optical density that represented viablecells was determined by the CellTiter 96 Aqueous Non-Radioactive CellProliferation Assay (Promega, Madison, Wis.). Cytotoxicity wascalculated as the percentage of control untreated cultures, mean±SD.n=3. MJDB was shown to be equally cytotoxic towards B lymphoma cellsexpressing either wild type or mutant p53, suggesting the potentialclinical use of MJDB against drug resistant tumors. See FIG. 3.

Example 4 MJDB Lowers ATP Levels in Cancer Cells

An ATP determination assay was used to assess the effect of MJDB on ATPlevels in cancer cells. More specifically, Molt-4 cells (at 1×10⁴/well)were seeded in 96-well opaque-walled plates and MJ at 3 mM or MJDB at 1mM were added for 10, 30 or 60 minutes at 37° C. Untreated cellsincubated in culture medium were used as control. ATT levels weredetermined by the CellTiter-Glo™ Luminescent Cell Viability Assay; anassay in which cells produce a lumineascence signal that is equivalentto the ATP concentration in the cell. ATP depletion is calculated as %of control untreated cultures, mean±SD. n=3.

As can be seen in FIG. 4, MJDB induced a rapid and strong ATP depletionin Molt-4 leukemia cells, to an extent bigger than that induced bymethyl jasmonate. It is important to note that MJDB was administered ata 3 times lower concentration than methyl jasmonate. Thus, a correlationexists between the superior cytotoxic effect of MJDB (in comparison tomethyl jasmonate) and its ability to reduce the cellular ATP levels.

Example 5 MJTB is Cytologic Against Four Diverse Human Malignant CellLines

Molt-4 (leukemia, at 2.5×10⁴/ml), D122 (lung carcinoma, at 5×10³/ml),B16 (melanoma, at 2×10³/ml), and B16MDR (melanoma exhibiting multidrugresistance, at 2×10³/ml) cells were incubated for one day in 96-wellplates in the presence of different concentrations of MJTB. The opticaldensity that represented viable cells was determined by the CellTiter 96Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison,Wis.), as described above. Cytotoxicity was calculated as % of controluntreated cultures, mean±SE. n=3. The IC50 levels of MJTB against thesecell lines are shown in the table below:

B16 Molt-4 D122 B16 MDR IC50 0.008 0.05 0.08 0.08 (mM)

As can be seen from this table and FIG. 5, MJTB was effective againstboth leukemic cells as well as cancer cells derived from various solidtumors. In addition, MJTB was capable of killing cells that expressmultidrug resistance, making it potentially useful in clinicalsituations where drug resistance is a major obstacle to successfulchemotherapy.

Example 6 Comparison of MJ and MJTB

Biological assays were performed with a 1:1 mixture of theaforementioned tetra bromo isomers. More specifically, Molt-4 leukemiccells (at 2.5×10⁴/ml) were incubated for one day in 96-well plates inthe presence of different concentrations of either MJ or MJTB. Theoptical density that represented viable cells was determined by theCellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay (Promega,Madison, Wis.) as described above. Cytotoxicity is calculated as % ofcontrol untreated cultures, mean±SE. n=3. The IC50 levels of MJTB and MJare shown in the table below:

MJTB MJ IC50 0.008 0.5 (mM)

As can be seen in this table and FIG. 5, MJTB has an IC50 (concentrationexhibiting 50% cytotoxicity) that is about 60 times smaller than that ofMJ (the most potent jasmonate derivative until now). Thus, MJTB is moreeffective than MJ by more than an order of magnitude.

Example 7 MJTB is Selective Against Cancer Cells

Molt-4 (leukemia) cells (at 2.5×10⁴/ml), and normal peripheral bloodlymphocytes (PBL, at 2×10⁵/ml) were incubated in 96-well plates for oneday in the presence of different concentrations of MJTB. PBL werepre-incubated with 0.8 μg/ml phytohaemagglutinin +5 ng/ml TPA for 48hours, to induce entrance into the cell cycle. These cells proliferateand therefore are similar in that respect to cancer cells, making thecomparison more valid. The optical density that represented viable cellswas determined by the CellTiter 96 Aqueous Non-Radioactive CellProliferation Assay (Promega, Madison, Wis.), as described above. IC50levels are shown in the table below:

Molt-4 PBL IC50 0.008 0.25 (mM)

As can be seen in this table and FIG. 7, there is a comfortabletherapeutic window which allows MJTB to kill leukemic cells withoutexerting a substantial effect on normal lymphocytes. Indeed, the IC50for normal peripheral blood lymphocytes is larger than that for leukemiccells by more than an order of magnitude. It should be noted that theIC50 of PBL was determined using higher MJTB concentrations that do notappear in FIG. 7.

Example 8 Cytotoxicity of Other Compounds of the Invention

Molt4 leukemic cells (at 1.5×10⁴/ml) were incubated for 1 day in thepresence of different concentrations of halogenated derivatives of MJ.Specifically, these derivatives were MJS72f5 (one of R₆ and R₇ is iodoand the other is methoxy), MJS99 (R₆ and R₇ are each fluoro) and NJ-63(one of R₆ and R₇ is bromo and the other is hydroxyl). The opticaldensity that represented viable cells was determined by the CellTiter 96Aqueous Non-Radioactive Cell Proliferation Assay (Promega, Madison,Wis.), described above. As shown in FIG. 8, all of these derivativesshowed greater cytotoxicity than MJ. Another derivative. MJS81f3, alsohad cytotoxic activity.

All references cited herein are incorporated in their entirety. It isappreciated that the detailed description herein above is intended onlyto illustrate certain preferred embodiments of the present invention. Itis in no way intended to limit the scope of the inventions as set out inthe claims that follow.

1. A compound of Formula I:

wherein: n is 0; R₁ is OH, C₁ to C₁₂ alkoxy, or C₁ to C₁₂ substitutedalkoxy; R₂ is oxo; R₃, R₄, R₅, C, D and E are each H; A and B are each Hor halogen; R₆ and R₇ are each selected from the group consisting offluoro, chloro, bromo, iodo, hydroxy, C₁ to C₁₂ alkoxy, and C₁ to C₁₂substituted alkoxy, provided that at least one of R₆ and R₇ is bromo,fluoro, chloro or iodo; wherein the bonds between C₃:C₇, C₄:C₅, andC₉:C₁₀ may independently be double bonds or single bonds; includingsalts, hydrates, optical isomers, enantiomers, diastereomers, andmixtures thereof.
 2. The compound of claim 1, wherein (a) R₆ and R₇ areeach bromo; or (b) R₆ and R₇ are each fluoro; or (c) R₆ and R₇ are eachiodo; or (d) R₆ and R₇ are each chloro; or (e) one of R₆ and R₇ is bromoand the other is hydroxy, C₁ to C₁₂ alkoxy, or C₁ to C₁₂ substitutedalkoxy; or (f) one of R₆ and R₇ is iodo and the other is methoxy.
 3. Thecompound of claim 1, wherein the bond between C₉: C₁₀ is a single bond.4. The compound of claim 3, wherein the bonds between C₃:C₇, C₄:C₅, andC₉:C₁₀ are single bonds.
 5. The compound of claim 1, wherein the bondbetween C₃:C₇ is a single bond.
 6. The compound of claim 1, wherein thebond between C₃:C₇ is a double bond.
 7. The compound of claim 1, whereinR₁ is OH, methoxy, t-butoxy or substituted alkoxy.
 8. The compound ofclaim 1, wherein: the bonds between C₃:C₇, C₄:C₅, and C₉:C₁₀ are singlebonds; R₁ is methoxy; A and B are each H; and R₆ and R₇ are each fluoro.9. The compound of claim 1, wherein: the bonds between C₃:C₇, C₄:C₅, andC₉:C₁₀ are single bonds; R₁ is methoxy; A and B are each H; and R₆ andR₇ are each iodo.
 10. The compound of claim 1, wherein: the bondsbetween C₃:C₇, C₄:C₅, and C₉:C₁₀ are single bonds; R₁ is methoxy; A andB are each H; and R₆ and R₇ are each chloro.
 11. The compound of claim1, wherein: the bonds between C₃:C₇, C₄:C₅, and C₉:C₁₀ are single bonds;R₁ is methoxy; A and B are each H; R₆ is hydroxy and R₇ is bromo. 12.The compound of claim 1, wherein: the bonds between C₃:C₇, C₄:C₅, andC₉:C₁₀ are single bonds; R₁ is methoxy; A and B are each H; one of R₆and R₇ is iodo and the other is methoxy.
 13. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier, and as anactive ingredient a compound of claim
 1. 14. The pharmaceuticalcomposition according to claim 13, wherein the composition is in a formsuitable for topical administration.
 15. The pharmaceutical compositionaccording to claim 14, wherein the composition is in the form selectedfrom the group consisting of an ointment, a cream, a gel, and drops. 16.A method for reduction of the growth of cancer cells, comprisingexposing the cancer cells to a therapeutically effective amount of acompound of claim
 1. 17. The method of claim 16, wherein the cancer is amammalian cancer.
 18. The method of claim 17, wherein the cancer is ahuman cancer.
 19. The method of claim 16, wherein the cancer is selectedfrom the group consisting of carcinoma, sarcoma, adenoma, hepatocellularcarcinoma, hepatoblastoma, rhabdomyosarcoma, esophageal carcinoma,thyroid carcinoma, ganglioblastoma, fibrosarcoma, myxosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphagiosarcoma, synovioama, Ewing's tumor,leimyosarcoma, rhabdotheliosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hematoma,bile duct carcinoma, melanoma, choriocarcinoma, seminoma, embryonalcarcinoma, Wilms'tumor, cervical cancer, testicular tumor, lungcarcinoma, small cell and non-small cell lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocyoma, medulloblastoma,craniopharyngioma, ependynoma, pinealoma, retinoblastoma, rectalcarcinoma, cancer of the thyroid, head and neck cancer, brain cancer,cancer of the peripheral nervous system, cancer of the central nervoussystem, neuroblastoma, cancer of the endometrium, lymphoproliferativediseases, hematopoietic malignancies including all types of leukemia andlymphoma including: acute myelogenous leukemia, acute myelocyticleukemia, acute lymphocytic leukemia, chronic myelogenous leukemia,chronic lymphocytic leukemia, mast cell leukemia, multiple myeloma,myeloid lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma.
 20. Amethod for the treatment of cancer comprising administering to thesubject in need thereof a pharmaceutical composition containing as anactive ingredient a therapeutically effective amount of the compoundaccording to claim
 1. 21. The method of claim 20, wherein the cancer isa mammalian cancer.
 22. The method of claim 21, wherein the cancer is ahuman cancer.
 23. The method according to claim 20, wherein the canceris selected from the group consisting of carcinoma, sarcoma, adenoma,hepatocellular carcinoma, hepatoblastoma, rhabdomyosarcoma, esophagealcarcinoma, thyroid carcinoma, ganglioblastoma, fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphagiosarcoma, synovioama, Ewing'stumor, leimyosarcoma, rhabdotheliosarcoma, colon carcinoma, pancreaticcancer, breast cancer, ovarian cancer, prostate cancer, squamous cellcarcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma,hematoma, bile duct carcinoma, melanoma, choriocarcinoma, seminoma,embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor,lung carcinoma, small cell and non-small cell lung carcinoma, bladdercarcinoma, epithelial carcinoma, glioma, astrocyoma, medulloblastoma,craniopharyngioma, ependynoma, pinealoma, retinoblastoma, rectalcarcinoma, cancer of the thyroid, head and neck cancer, brain cancer,cancer of the peripheral nervous system, cancer of the central nervoussystem, neuroblastoma, cancer of the endometrium, lymphoproliferative ,hematopoietic malignancies including all types of leukemia and lymphomaincluding: acute myelogenous leukemia, acute myelocytic leukemia, acutelymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocyticleukemia, mast cell leukemia, multiple myeloma, myeloid lymphoma,Hodgkin's lymphoma, non-Hodgkin's lymphoma, as well as metastasis of allthe above.
 24. The method of claim 20, wherein the cancer is selectedfrom the group consisting of prostate cancer, breast cancer, skincancer, colon cancer, lung cancer, pancreatic cancer, lymphoma,leukemia, head and neck cancer, kidney cancer, ovarian cancer, bonecancer, liver cancer, melanoma and thyroid cancer.
 25. The methodaccording to claim 20, wherein the composition is in a form suitable foradministration via a route selected from the group consisting oftopical, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, ophthalmic, and oral.